DNA expansion is a highly desired process that may benefit and enable several next generation DNA sequencing approaches; however, a practical and inexpensive DNA expansion process is currently not available. DNA expansion is a physical process in which the information of single nucleotides is translated into a much larger and easily readable structure (called a reporter). Nanopores are ideal readers for the proposed expanded constructs (called Xpandomers). In this proposal, Stratos Genomics and the University of Washington are merging strengths and partnering to develop and test expanded DNA. In Stratos Genomics' DNA expansion process, four-nucleotide long probes (tetramer Xprobes) are sequentially ligated along a target DNA in a novel ligation process. A selectively cleavable bond connects the 2nd and 3rd nucleotides of the Xprobe. The two halves of each Xprobe are also connected via a long tether molecule. These tethers are user-defined molecules (e.g. DNA sections) that comprise reporters for encoding the four nucleotides of the Xprobe. Breaking of the cleavable bonds enables expansion of the polymer backbone. The resulting construct is called an Xpandomer. To date we have: (i) Demonstrated synthesis of the Xprobes; (ii) Demonstrated synthesis of Xpandomers; (without reporters) by the solution-based template-dependant extension of a primer using a novel ligation process; (iii) Demonstrated high ligation specificity and processivity; (iv) Fabricated very low noise solid-state SiNx nanopores; (v) Synthesized and measured high signal nanopore reporters with very low noise SiNx nanopores; (vi) Demonstrated Duplex Interrupt (DI) nanopore sequencing using the pore protein MspA (UofW); (vii) Sequenced DNA constructs mimicking expanded DNA in a MspA nanopore. The crucial steps in our project will be to optimize the DNA expansion process and to prove sequencing with the protein pore MspA and with solid-state nanopores. In parallel we will develop short (~17 base) DI-reporter sequences to be read in MspA and long (~100-base) reporters to be read in SiNx pores. Our two-year goal is to demonstrate conversion of 32-base DNA into Xpandomers and read them with MspA or solid-state nanopores. Specific aims: i) Survey and select optimal ligase for ligation of tetramer Xprobes, ii) Synthesize 32-base encoded Xpandomers with full library of tetramer DI-Xprobes, iii) Sequence 32-base encoded Xpandomers in an MspA nanopore, iv) Develop a 4-state reporter for solid-state Xprobes and v) Sequence 32-base encoded Xpandomer in a solid-state SiNx nanopore.